Microbial modification of benzodiazepine compounds

ABSTRACT

METHOD FOR MICROBIAL TRANSFORMATION OF ABENZODIAZE PINE COMPOUND OF THE PARTIAL FORMULA   &gt;N-CO-CH2-N=C&lt;   TO A BENZODIAZEPINE COMPOUND OF THE PARTIAL FORMULA   &gt;N-CO-CH(-OH)-N=C&lt;   BY FERMENTING THE COMPOUND OF PARTIAL FORMULA I IN THE PRESENCE OF A STRAIN OF STREPTOMYCES, OR BY INCUBATING THE COMPOUND OF PARTIAL FORMULA I WITH RESTING CELLS OF A STRAIN OF STREPTOMYCES PREVIOUSLY GROWN IN A CULTIRE MEDIUM.

United States Patent U.S. Cl. 195--51 R 24 Claims ABSTRACT OF THE DISCLOSURE Method for microbial transformation of a benzodiazepine compound of the partial formula by fermenting the compound of partial Formula I in the presence of a strain of Streptomyces, or by incubating the compound of partial Formula I with resting cells of a strain of Streptomyces previously grown in a culture medium.

The present invention pertains to a method of causing the microbial transformation of a benzodiazepine compound having the following general Formula I:

wherein R is selected from the group consisting of hydrogen, carbamoyl, C -C alkylcarbamoyl, C -C alkyl, C -C alkenyl, C -C alkynyl, and C -C alkyl, C C alkenyl and C -C alkynyl substituted by one to three radicals selected from the group consisting of halogen,

cyclo C -C alkyl, aryl, heterocyclic ring, aralkyl, acyl, carbamoyl, C -C alkylcarbamoyl, amino, C -C alkylamino, di C -C alkylamino and --X-R (wherein X is one of the group consisting of oxygen, sulfur, sulfonyl and sulfinyl, and R is one of the group consisting of hydrogen, C -C alkyl, C -C alkoxy, C -C alkyl, halo C -C alkyl, C -C alkenyl, C -C alkynyl, aryl, acyl, aralkyl, heterocyclic ring, carbamoyl and C -C alkylcarbamoyl); R is selected from the group consisting of hydrogen, halogen and C1-C4 alkyl; R is selected from the group consisting of hydrogen, halogen, lower C -C alkyl and lower C -C alkoxy; R is selected from the group consisting of halogen, trifluoromethyl, C -C alkoxy, alkyl and nitro; R is selected from the group consisting of hydrogen, halogen, C -C alkyl and C -C alkoxy, and n is 0 orl, which method comprises fermentice ing a compound of Formula I above in the presence of a certain strain of Streptomyces to obtain a compound of the group having the following general Formula II:

wherein R R R R R and n have the same meaning as in the Formula I, or incubating a compound of Formula I with resting cells of Streptomyces previously grown in an adequate medium to obtain a compound of Formula II.

More particularly a certain species of Streptomyces is cultured in an adequate medium preferably under aerobic conditions and either from the beginning of growth or after growth a benzodiazepine compound of the Formula I is added to the broth and transformed to a compound of the general Formula II.

On the other hand the microbial modification of such benzodiazepine compounds is also carried out by the method of using resting cells previously grown on an adequate medium containing a benzodiazepine compound of the general Formula I.

The products are isolated from fermented or incubated fluids by extracting with organic solvents. The compounds obtained by the method of the present invention are generally optically active and useful as intermediates for other benzodiazepines and/or also as tranquilizing agents.

The compounds having general Formula I can be manufactured by chemical methods such as described in Japanese patent application No. 45 28,187.

Such compounds also can be produced from the compounds having general Formula I, where R is hydrogen, by treating the latter with active esters of alcohols having general formula R -OH, wherein R has the same meaning as in the Formula I.

The composition of the nutrient medium utilized for culturing the microorganisms, as well as for the fermentation, may vary widely.

The microorganisms suitably employed in the process of the present invention are conveniently grown in a nutrient composition suitable for the proliferation of the cells which produce the hydroxylating enzyme.

Generally speaking, such nutrient medium should contain assimilable sources of nitrogen and carbon, and sources of such other nutrients as phosphorous, sulfur, potassium, trace elements and such other growth-promoting factors as the microorganisms may require.

As sources of nitrogen the following can be used: soy bean meal, peptone, meat extracts, corn steep liquor, asparagiuc, urea and mixtures thereof. As sources of carbon: glucose, starch, glycerol, dextrin, molasses and combinations thereof are satisfactory.

The pH of the culture medium and fermentation systems may preferably be near 7.0.

The temperature of these systems may vary from about 20 C. to about 37 C., preferred range being from about 28 C. to about 30 C.

The fermentation is aerobic and, moreover, agitation and aeration are desirable for optimum results.

Fermentation is conducted in an aqueous medium described above in an aerated fermentor. A strain selected from Streptomyces is inoculated and cultured. Either after growth or at the beginning of growth the substrate shown by Formula I is added and its substantial converof benzodiazepine compound of the Formula I at R as well as hydroxylation in Formula I, wherein R is of the group consisting of various radicals except hydrogen, may be effected.

H sron to the compound shown in Formula II rs usually ef- 5 fected during a 2 to 8 day fermentation period. /NR! /N-H The substrate concentration may be varied over a wide H range up to g. or more per liter of culture being used.

It is preferred, however, to operate in the range of 10 Thlsmventlon also1I191l1d$th1Rr0C6SS 5 5 grams f substrate liter of culture The novel conversion of this invention can be accom- The substrate may be added with or without dilution phshedsby y Strains hQ h il i sls lccted from in a suitable solvent. Perferably, it is added as a 5-10 genus treptcmlfces W ave y Y g enzyme percent solution in an organic solvent such as ethanol. h Suc h mlcroorgamsms eXemPhfied y the T he substrate may be contacted with the microorganism g speclesh p x%gg 3 g g ATCC and its enzymes without removing them from the growth treptomyces treptomyces gr 1880- medium or, alternatively the microorganism cells and enigg gg6 g g 14511, i ;i; gg g m zymes may be first separated from the growth medium genes I p myces ra lae treptoand contacted with the substrate in a suitable butter other wf' tg i g g 2 p y z g g g o. tre tom ces ca vus 'l h su b tfa t t e i t he l ormula, I may be added all at SlrePmmDCeS, fimifariusyslz PERM-P once to the growth culture or enzyme preparation, in Streptomyces charfreusis PERM-P 946, p portions, or continuously during the growth and progress "W l gff g l io lgE gii gg 3 48 21 g p of th ti myces g o zsporus 0. an trepto- The contact of the substrate of the Formula I with mi- 25 myves benzvdlwzepmicus -1 5 PERM-P I I0. 949. croorganism or enzymes is effectively accomplished by The f' stfalns have been lsolatedby addition of fine suspension of substrate made by super- PWSBIIt fmm $011 have been deP0$1ted 111 soni tr atm t, the Fermentat1on Research Institute Agency of Industrial The products obtained by the microbial conversion, Science and Tg gy, ChIba, p under h above i.e., the compounds of Formula II, can be isolated from a CSSI I Hum ers the reaction mixture by conventional techniques utilizing The names of PERM-P strainsw re s g y differences between the products and impurities; such the results of the fOllOWlng classification for Which Waksproperties as solubility, adsorbability, distribution coei-fiman System (S. A. Waksman, The Actinomycetes, vol. II, cient between solvents, etc. Culture fluids are extracted 1961) was adopted. with organic solvents such as chloroform, carbontetra- These strams form spores and aerial myceha, t d chloride, ethylene dichloride, dichloromethane, ether, etc., not form sporangra. and concentrated. Vegrtatrve mycelrum 1s nonseptate and not fragment ng The concentrates obtained may be further purified, if rnltlo bztrfirllary or cogoid elemenlgs. Spores Sform ll'l chalns. desired, by various techniques such as column or thin T us ese strains e ong to t e genus treptomyces in layer chromatography using silica gel or sephadex, extrac- 40 Family II Streptomycetaceae. tion with acids and recrystallization. The details of taxonomic studies of these strains are Under certain conditions the process of dealkylation shown in the following tables.

TABLE I Morphology of Branched,intormoivertlcilis,long Branched, no vertlcillate, straight or Branched, short, straight.

sporophores. slightly wavy with loose spirals. Number of spores..- More than ten formed in chains More than ten formed in chains. More than ten formed in chains. Shape of spores-.-" Polyhedral 0.5X0.9p Cylindrical 0.5-0.6XCL9-1Au Pollyhedral or cylindrical 0.60.7X1.1-

. t. Growth on solid medium:

Sucrose-nitrate Growth moderate, gray to cream; colony Growth poor or moderate, white to gray; Growth good, white to dull-yellow; agar. flaltit aterial mycelium produced late, aerial mycelium produced late, white to firgggtedpggtlierybuaegizallt mylcietleiuirg w e ogray. g y- Y1 W ello ish Glucose- Growth moderate, gray to cream; colony Growth moderate, gray to white; colony Gr t iwtli mogsi te yellowish-gray; aerial asparaglne clrifillal'; aerial mycelium abundant, circular; aerial mycelium good, white to igngcelium produced, moderate, yellowa S a stgg h agar....- G wti r good, gray or white to dark Gr tiw th good, gray to brownish gray; Grow l r good, white to dull-orange;

yellowish-brown; aerial mycelium abundant, white to gray.

Tyrosine agar..- Growth moderate or poor, white to yellowish-orange; colony circular, cottony; aerial mycelium produced early, abundant, white.

Nutrient agar..- Growth moderate, white or yellow; colony circular rigid; aerial mycelium produced early, abundant white.

Potato-glucose Growth good, white to purple to brownagar. ish-black; colony circular, ngid; aerial mycelium produced early, abundant,

gray or pinkish-white. Pepton-glucose Growth good, gray to brown; colony agar. spindly raised, rugous; aerial mycclium produced late, poor, gray.

colony flat; aerial mycelium abundant,

white to Growth poor, gray or brown; colony flat;

aerial mycelium poor, white or gray.

Growth good, gray or brown; colony spindly, raised, rough surface; aerial mycelium produced early, poor, white gray.

aerial mycelium produced abundant, pale yellowish-brown. Growth good, gray to pale yellowishbrown; aerial mycelium produced early, abundant, yellowish-gray.

Growth good white to brownish-yellow; aerial myce iurn produced early, white to yellowish-gray.

Growth abundant, white or gray to brown; colony powdery, raised; aerial mycelimn produced early, abundant, yellowish-gray.

Growth good, white or gray; colony rigid, surface flat; aerial mycilium produced early, abundant, gray; or

early,

flat; aerial mycelimn abunor gray. yellowish-gray. Loeflier's serum. Growth moderate, white, membranous-.-. Growth moderate, white or gray Growth good, white or gray; aerial mycelium, pale yellow.

Morphology of Monopodially branched, with Monopodially branched with Branched in form of tufts, Branched, verticilate, loop sporophores. screw coils. sh rt oils straight or slightly curved.

shape o c Number oi spores... More than ten formed in chains- More than two formed in chains. More than ten formed in More than ten formed in chains.

surface covered with Polyhedra10.80.9 1.0

chains. 4.4 4- Cylindrical 0-6-0-8X1.01.5p Round to oval 0.9-1 -3X1.Q-1.5p-

TABLE IContinued S-19 S450 S420 S445 Growzih on solid me um:

Sucrose-nitrate Growth good, greenish-gray; Growth good, gray to yellow- Growth moderate, dull- Growth poor, whitetograyishagar. aerial mycelium produced ish-brown; aerial mycelium orange-bud; aerial mycelibrown; aerial mycelium proearly, abundant, bluish-gray. produced late, abundant, um produced early, good, duced early grayish-red to bluish-gray. yellowish-gray. brown.

Glucose- Growth good, colorless; colony Growth good, dark brown; Growth moderate, pale yel- Growth poor to moderate,

asparagine wet, yeast-like; aerial mycelicolony raised; aerial mycelilow; colony wet, circular; light bluish-gray; aerial myegar. um produced late, bluishum produced early, abunaerialmycelium poor, white cehum produced early, abungray. dant, bluish-gray. or pale yellow. dant, grayish-red brown.

Starch agar Growth good, gray to brown- Growth good, white or pale Growth good, white to pale Growth good, white to grayish gray; aerial mycelium cream; aerial mycelium proyellowish-brown; aerial myish-brown; colony white,

produced early, abundant, duced early, abundant, gray cehum produced early, cottony; aerial mycelium gray or bluish-gray. to brownish-gray. abundant. alpundant, grayish-red rown Tyrosine agar... Growth good, gray black;

aerial mycelium produced early, abundant, brownishrownish-gray or colorless; aerial mycelium scanty.

Growth poor; colony gray; aerial mycelium produced early, gray.

Growth moderate or poor, colorless or gray; colony circular, flat; no aerial mycelium.

Growth good, gray to yellowish-gray; colony fiat, circular; aerial mycelium abundant, gray or yellowish-gray.

Growth good, white to light brown; colony circular, fiat; aerial mycelium produced early, abundant, white or yellowish-gray.

Growth abundant, white to Growth poor, white to grayishred brown; aerial mycelium grayish-red brown.

Growth moderate or poor, gray or white to yellowishg ray; colony wet, bacteriahke; no aerial mycelium.

tatolucose Growth ood yellowish white Growth good gray or bluish Growth abundant white to i g to dark bluish-green; colony gray; colony rugous, raised; orange to dark bluish-gray; yellowish-browm'colony cotwet, rugous, yeastlike; aerial mycelium abundant, aerial mycehum produced tony; aerial mycelium proaerial mycelium produced bluish-gray orgray. late, abundant, pale yellowduced early, grayish-brown. litahabundant, gray or ish-brown.

111$ -gray.

Peptone- Growth moderate, gray to yel- Growth good, pale yeilowish- Growth good, yellow to Growth good, white to Dal glucose lowish-gray to dark brown; brown; colony raised, rugous; brown;aerialmycel1um pnr yellow;aerlalmycehumpoo agar. colony yeast-like, wet, ruilae'ial mycelium produced ducedearly,yellowish-gray.

ous. a e, gray. Loefilers Gr owth good, yellowish-gray; Growth moderate or poor, Growth good, white, mern- Growth poor, white or gray.

serum. aerialmyceliumyellow. white. briiitious; aerial mycehum TABLE II [Physiological condition] S-3 S-4 S-12 timum tem erature C. 25-37; optimum a 30 25-37 op imum a 30 25-40; optimum at 37. 1531mm to pIE I 1-3 pH 6-10; optimum at pH 8 pH 6-10; optimum at pH 8. pH 6-10; optimum at pH 3, Liquefaction of gelatin N None Liquefaction Action on ilk Slight coagulation; no peptonization.-. No coagulation; peptonization-..-. No coagulation; peptonization, Melanin formation. N N None. Hydrolysis of starch Positiv Positiv Positive. Action on nitrate... Nitr N No nitrite.

S-19 S-60 S-i20 S-145 0 timum tem erature 0... -40; optimum at 37 25-37 optimum at 25-40; optimum at 30 25-40; optimum at 37. R lation to pHE pH 6-10; optimum at pH 8.. pH 4-10; optimum at pH 6.- phi 6-10; optimum at pH 8.. pH 4-10; optimum at pH 7. Liquefaction of gelatin .im'lofnoflnn None Liquefaction None. Action on milk No coagulation; peptoniza- Strong coagulation; no pep- N c agulation, peptomza- Coagulation, peptonizatlon.

tion. tonizatron. tron. Melanin formation Melanin formed Melanin formed None None. Hydrolysis of starch Positiv Posit ve Positive Positive. Action on nitrate.... Nitri No m rlf Nirnra No mtrite.

(2) S-4 ('FERM-P No. 944): Among known species, stram S-4 has many common characteristics to Strepto- TABLE III [s a 81mm mm] myces calvus Backus et al., 1957, but differs from it in 5 such properties as partial liquefaction of gelatin coagu- S42 S49 5-120 3-145 lation of milk, and the pattern of assimilation of rafiinose t e l r i i i i $13??? S... 1 d

-xy ose us e s am is to er am ne 0 a n w strain D-glucose.-. f St I l P P y g t e Diructose" 0 rep omyces ca v,us. ISucSrfisel.-- I i i i i i (3) S-l2 (FERM-P No. 945): In respect of many t?h ,,;;.;gg 6 characteristics, strain S-l2 resembles to Streptomyces Baffinose...-- micarium Waksman and Henrici 1948 diffe D-mannitole :i: fi but rs from it in such properties as shape of spore and mode of growth on nutrient agar. Thus it is thought to be proper to assign strain S-l2 to a new strain of Streptomyces fimicarium.

(4) S-19 (PERM-P No. 946): Strain S19 does not produce soluble pigments on starch media, nutrient agar media, and potato agar media. Besides the production of soluble pigments, other characteristics of strain S-19 also resembles to Streptomyces chartreusis Calhoun and Johnson, 1956. Thus the strain S-19 is properly assigned to a new strain of Streptomyces chartreusis S-l9.

(5) 5-60 (PERM-P No. 947): Spores are formed in sporophores which arise from the aerial mycelium in the form of verticils. Melanin is produced on tyrosine agar media.

Brown growth is obtained and aerial mycelium is white or gray.

From these results, the strain -60 resembles to Streptomyces griseochromogenes Fukunaga et al., 1955. HOW ever, both strains difier in the following properties: the color of growth and aerial mycelium on sucrose agar media, the production of aerial mycelium on asparagine agar media, the production of aerial mycelium, color of soluble pigments on nutrient agar media, the liquefaction of gelatin, the coagulation and the peptonization of milk, and the resuction of nitrate.

From these resutls the strain 8-60 is assigned to a new species and named Streptomyces transformans S-60.

(6) S-120 (FERM-P No. 948): Strain 8-120 has many common characteristics to Streptomyces globispams Waksman among known species. However, strain S-120 does not produce soluble pigments on sucrose agar media and glucose-peptone agar media, on which growth is colorless or greenish. From these points the strain S-l20 is seemed to be a new strain of Streptomyces globisporus.

(7) 8-145 (FERM-P No. 949): Sporophores arise from aerial mycelium in the fom of verticils and form compact loops. Melanin is not produced, growth is yellowish or gray and aerial mycelium is white or pinkish. Among known species, Streptomyces hachijoensis Yamaguchi, 1954 and Streptomyces matensis Margalith et al., 1959 have relatively resembling characteristics to the strain S-145.

However, Streptomyces hachijoensis shows White or pinkish-buff growth on sucrose agar media, forms cottony aerial mycelium on nutrient agar media and rapidly liquefiies gelatin. On the other hand, Streptomyces matensis produces bluish soluble pigments on sucrose agar media and forms white aerial mycelium on nutrient agar media.

From these points it is difficult to assign the strain S-145 to either of the two described above. Therefore the strain S-145 was designated as a new species and named Streptomyces benzodiazepinicus 8-145.

According to the present process, the following benzodiazepine compounds are produced, for example;

3-hydroxy-5-phenyl-7-chl0ro-1,3-dihydro-2H-1,4-

benzodiazepine-Z-one,

3 -hydroxy-5-phenyl-7-nitro-1,3-dihydro-2H-1 ,4-

benzodiazepine-Z-one,

3 -hydroxy-5-phenyl-7-trifluoromethyl-1,3-dihydro- 2H-1,4-benzodiazepine-2-one,

3-hydroxy-5-phenyl-7-methoxy-1,3-dihydr0-2H-1,4-

benzodiazepine-Z-one,

3-hydroxy-5-phenyl-7-methyl-1,3-dihydro-2H-l,4-

benzodiazepine-Z-one,

3-hydroxy-5-phenyl-7,9-dichloro-1,3-dihydro-2H-1,4-

benzodiazepine-Z-one,

3-hydroxy-5-(o-chlorophenyl) -7-chloro-1,3-dihydro- 2H-1,4-benzodiazepine-2-one,

3-hydroxy-5- (o-chlorophenyl -7-nitro 1,3-dihydro- 2H-1,4-benzodiazepine-2-one,

S-hydroxy-S- (o-fiuorophenyl -7-chloro-1,3-dihydro- 2H-1,4-benzodiazepine-2-one,

3-hydroxy-5-(o-fiuorophenyl) -7-nitro1,3-dihydro- 2H-1,4-benzodiazepine-2-one,

3-hydroxy-5-(o-tolyl) -7-chloro-1,3-dihydro-2H-1,4-

benzodiazepine-Z-one,

3-hydroxy-5- p-hy droxyphenyl -7-ch10ro-1, 3-dihydro- 2H-1,4-benzodiazepine-2-one,

3-hydroxy-5-(p-chlorophenyl)-7-chloro-1,3-dihydro- 2H-1,4-benzodiazepine-2-one,

3-hydroxy-5- (o-chloro-p-hydr oxy-phenyl) -7-chloro- 1 ,3

dihydro-ZH- 1 ,4-benzodiazepine-2-one,

3-hydroXy-5- (o-methyl-p-chloro-phenyl) -7-chloro- 1, 3-

dihydro-ZH-1,4-benzodiazepine-2-one,

1-methyl-3-hydroxy-5-phenyl-7-chloro-1,3 -dihydro- 2-H-1,4-benzodiazepine-2-one,

1-methyl-3 -l1ydroXy-5-(o-chloro-phenyl)-7-chlor0-1, 3-

dihydro-1,4-benzodiazepine2-one,

l-methy1-3 -hydroxy- 5- (o-fluoro-phenyl) -7- chloro-1 3- dihydro-ZH-1,4-benzodiazepine-2-one,

l-methyl-3-hydroxy-S- o-chloro-phenyl -7-m'tro-1,3

dihydro-ZH-1,4-benzodiazepine-2-one,

l-methyl-3-hydroxy-5-phenyl-7-nitro-1, 3-dihydro-2H- benzodiazepine-Z-one,

1-methyl-3 -hydroxy-S-phenyl-7-trifiuoromethyl-1,3

dihydro-ZH-1,4-benzodiazepine-2-one,

1-cyclopropylmethyl-3-hydroXy-5 -phenyl-7-chloro-1 ,3-

dihydro-ZH-1,4-benzodiazepine-2-one,

1 -cyclopropylrnethyl-3 -hydroxy-5-(o-ch1oro-phenyl)-7- chloro- 1,3-dihydro-2H-1,4-benzodiazepin-2-one,

1-methylcarbamoyl-3-hydroxy-5-phenyl-7-ch1oro- 1 ,3-

dihydro-2H-1,4-benzodiazepine-2-one,

1-metl1ylcarbamoyl-3-hydroxy-5-phenyl-7 -nitro1-1 ,3-

dihydro-ZH- 1 ,4-benzodiazepine-2-one,

l-methylcarbamoylmethyl-3-hydroxy-5-phenyl-7-chloro- 1,3-dihydro-2H-1,4-benzodiazepine-2-one,

1- B-ethylaminoethyl -3-hydroxy-5- (o-fluorophenyl) -7- chloro-1,3-dihydro-2H-1,4-benzodiazepine-2-one,

1- (fi-diethylaminoethyl -3-hydroxy-5-phenyl-1- (ofiuorophenyl)-7-chloro-1,3-dihydro-2H-1,4-

benzodiazepine-Z-one,

1- ('y-dimethylaminopropyl -3-hydroxy-5- (o-fiuorophenyl -7-chloro- 1, 3 -dihydro-2H-,1,4-benzodiazepine-Z-one,

1- (fl-piperydinoethyl -3 -hydroxy-5- (o-fluorophenyl) -7- chloro-1,3-clihydro-2H-1,4-benzodiazepine-2-one,

1- (B-hydroxyethyl -3 -hydroxy-5- o-fiuorophenyl) -7- chloro 1 ,S-dihydro-ZH- 1 ,4-benzodiazepine-2-one,

1- (,B-ethoxyethyl -3 -hydroxy-5- (o-fluoropheny1)-7- chloro- 1,3 -dihydro-2H- l ,4-benzodiazepine-2-one,

1-methoxymethy1-3 -hydroxy-5-pheny1-7-chloro-1,3-

dihydro-ZH-1,4-benzodiazepine-2-one,

1-methoxymethyl-3 -hydroxy-5-phenyl-7-nitro-1,3-

dihydro-ZH-1,4-benzodiazepine-2-one,

1- B-ChlOIO-oz-IHBthOXY) -ethyl] -3 -hydroxy-5-phenyl-7- chloro-1,3-dihydro-2H-1,4-benzodiazepine-2-one,

1- (methylthiomethyl) -3-hydroxy-5-phenyl-7-ch1oro-1 ,3-

dihydro-ZH- 1 ,4-benzodiazepine-2-one,

1- methylsnlfinylmethyl -3-hydroxy-5- o-chlorophenyl) 7-chloro- 1, 3-dihydro2H- l ,4-benzodiazepine-2-one,

1- fi-methylsulfonylmethyl) -3 -hydroxy-5- (o-fluorophenyl -7-chloro-1,3-dihydro-2H-1,4- benzodiazepine-Z-one,

1- methoxyethoxyethyl -3-hydroxy-5 -phenyl-7-ch1oro- 1,3-dihydro-2H-1,4-benzodiaZepine-2-one,

1- fl-acetoxyl-methoxy ethyl] -3-hydroxy-5-phenyl-7- chloro-1,3-dihydro-2H-1,4-benzodiazepine-2-one,

1- fi-hydroxyll-methoxy) ethyl] -3 -hydroxy- 5 -pheny1- 7-chloro-1,3-dihydro-ZH-1,4-benzodiazepine-2-one,

1- B-phenoxy-ethyl -3 -hydroxy-5 -pheny17-chloro- 1,3-

dihydro-ZH-1,4-benzodiazepine-2-one,

1- B-benzyloxy-ethyl -3 -hydroxy-5-pheny1-7-chloro-1,3

dihydro-ZH-1,4-benzodiazepine-2-one,

1- B-pyranyloXy-ethyl) -3 -hydroxy-5- (o-fiuorophenyl) -7- chloro-1,3-dihydro-2H-1,4-benzodiazepine-2-one,

1- (fi-vinyloxy-ethyl) -3-hydroxy-5-phenyl-7-chloro-1,3-

dihydro-2H-1,4-benzodiazepine-2-one,

1- (,B-chloroethoxy) methyl-3 -hydroxy-5-phenyl-7-chloro- 1,3 -dihydro-2H-1,4-benzodiazepine-2-one, and

1- 5, 3, 8,-trifluoroethyl -3-hydroxy-5-phenyl-7-chloro- 1,3-dihydro-2H-1,4-benzodiazepine-2-one.

The present process is illustrated in greater details by the following examples, which are to be construed as illustrative and not limitative of the invention.

EXAMPLE 1 Streptomyces transformans S-60 (PERM-P No. 947) from an agar slant was transferred to a 500 ml. Sakaguchi flask containing ml. of the following medium; glucose 5 g., yeast extract 0.5 g., soy bean meal 0.5 g.,

9 KHP O 0.1 g., K HPO 0.2 g., FeSO -7H 0.1 g., tap water 100 ml., pH 7.0.

The flask Was incubated on a reciprocal shaker at 30 C.

After 24 hours of culture, 2% of a mycelial transfer was made to each of twenty new Sakaguchi flasks containing 100 ml. of the same medium as described above. After additional 24 hours of incubation as above, 100 mg. of l-cyclopropyl methyl-5-phenyl-7-chloro-1,3-dihydro- 2H-1,4-benzodiazepine-2-one in 2.0 ml. of ethanol was added to each flask. After further 7 days incubation culture fluids were extracted with chloroform and extracts were evaporated to dryness under reduced pressure.

The residue, 1.93 g., was dissolved in a mixture of chloroform/acetone/ethanol (17:2:1 by volume) and then applied on the top of the silica gel column. The development was carried out by the same solvent and 1- cyclopropylmethyl 3 hydroxy-5-phenyl-7-chloro-1,3- dihydro-ZH-1,4-benzodiazepine-2-one melting at 159- 161 C. and 3-hydroxy-5-phenyl-7-chloro-1,3-dihydro- 2H-1,4benzodiazepine-2-one melting at 203-204 C., [a] =+1O.4 (c.=0.5, in methanol) were obtained.

The infrared spectra of those products were identical with those of l-cyclopropylmethyl-3-hydroxy-5-phenyl-7- chloro-1,3-dihydro-2H-1,4benzodiazepine-2 one and 3- hydroxy 5 phenyl 7 chloro-1,3-dihydro-2H-1,4- benzodiazepine-Z-one, respectively.

EXAMPLE 2 Streptomyces benzodiaze inicus S1-45 (PERM-P No. 949) was grown on the same medium described as Example 1 at 28 C. After 24 hours incubation a mycelial transfer (2%) was made to each of thirty new Sakaguchi flasks containing 100 ml. of the same medium as above.

After good growth 100 mg. of 5-(2'-fluorophenyl)- 7 chloro 1,3 dihydro-ZH-1,4-benzodiazepine-2-one in 2.0 ml. ethanol was added to each flask. Following 5 days incubation, culture fluids were extracted with chloroform and 2.27 g. of crude crystals were obtained by the same procedure described in Example 1.

The crude crystals were dissolved in 20 ml. of chloroform and applied on Sephadex LH-20 column (2.5 cm. x 80 cm.). The substrate were eluted by chloroform, and

then 3 hydroxy 5 (2'-fluorophenyl)-7-chloro-1,3-

dihydro-ZH-1,4-benzodiazepine-2-one was obtained by elution with ethanol.

The residue derived from ethanol eluates was recrystallized from ethanol. The physicochemical properties of 3- hydroxy 5 (2 fluorophenyl)-7-chloro1,3-dihydro- 2H 1,4 benzodiazepine-Z-one obtained as above were as follows: M.P. 193-194 C., [u] =31.0 (c.=0.58, DMSO).

Elementary analysis.Calculated (percent): C, 59.13; H, 3.31; N, 9.19; CI, 11.63. Found (percent): C, 59.15; H, 3.36; N, 9.09; Cl, 11.41.

EXAMPLE 3 A mycerial transfer of Streptomyces calvus S-4 (FERM-P No. 944) previously grown on the same media as in Example 1 was made to each of twenty flasks containing ml. medium of the same composition as in Example 1. Following 48 hours cultivation at 28 C., 100 mg. of 1-methyl-5-phenyl-7-nitro-1,3-dihydro-2H-1,4 benzodiazepine-Z-one in 2.0 ml. of ethanol was added to each flask. After further 8 days cultivation culture fluids were extracted as described in Example 1.

The crude crystals were purified as the similar procedure in Example 2 by which yielded 1-methyl-3-hydroxy- 5-phenyl-7-nitro-1,3-dihydro-2H 1,4 benzodiazepine-2- one melting at 201-202 C. and 3-hydroxy-5-phenyl-7- nitro-1,3-dihydro-2H-1,4-benzodiazepine-2-one melting at 199-201 C.

The infrared spectra of these are identical with the ones of standard compounds.

EXAMPLE 5 The fermentation was conducted with Streptomyces fimicarius S-12 (PERM-P No. 945) as described in Example 1, except that the substrate l-methyl-5-phenyl-7- trifluoromethyl-1,3-dihydro 2H 1,4 benzodiazepine-Z- one was added at the beginning of the growth thereto.

The culture fluids were extracted with the method as in Example 1. The crude products were separated and purified as in Example 2, by which yielded 1-methyl-3- hydroxy-5-phenyl-7-trifluoromethyl 1,3 dihydro-2H-1,4- benzodiazepine-Z-one melting at 183-185" C. and 3-hydroxy-5-phenyl-7-trifluoromethyl 1,3 dihydro-2H-1,4- benzodiazepine-Z-one melting at 189191 C.

The identity of those compounds was respectively established by IR spectroscopy.

EXAMPLE 6 The fermentation was conducted with Streptomyces chartreusis S-19 (PERM-P No. 946) as described in Example 1, except that 2 ml. of sonicated suspension containing 100 mg. of substrate, 5-phenyl-7-chloro-l,3-dihydro-2H 1,4 benzodiazepine-2-one, was added to each flask. The fine suspension was made by sonication of substrate suspended in the medium having the same composition as in Example 1.

The extraction was carried out according to the procedure described in Example 1 and 1.97 g. of crude crystals were obtained.

The separation and purification of the crude products carried out as described in Example 2 which yielded 3-hydroxy-5-phenyl-7-ehloro-1,3-dihydro 2H 1,4-benzodiazepine-Z-one melting at 202-203 C.

The infrared spectrum of this compound was identical with the one of the standard compound.

EXAMPLE 7 Streptomyces globisporus S- (PERM-P No. 948) was grown in a 500 ml. Sakaguchi flask containing 100 ml. of the medium as described in Example 1.

Sixty ml. of the heavy growth which had developed was transferred to 6 liters of the medium described above in a 10 liter stirred jar fermentor. The fermentor was incubated at 30 C. and during fermentation an air flow and agitation were maintained.

After 24 hours cultivation 6 g. of 1-methyl-5-(2'-chlorophenyl)-7-chloro 1,3 dihydro-ZH-1,4-benzodiazepine- 2-one dissolved in ethanol was added thereto. Following 6 days cultivation extraction was carried out as described in Example 1 and 5.84 g. of crude crystals were obtained.

The separation and purification of the crude mixture were accomplished by the method described in Example 2 and yielded l-methyl-3-hydroxy-5(2-chlorophenyl)-7- chloro-1,3 dihydro-ZH-1,4-benzodiazepine-2-one melting at 197-200 C. and 3-hydroxy-5-(2'-chlorophenyl)-7- chloro-1,3-dihydro-2H 1,4 benzodiazepine-Z-one melting at -167 C.

The identity of those compounds was respectively established by IR spectroscopy.

1 1 EXAMPLE 8 A mycerial transfer of Strepfomyces benzodiazepinicus S-145 (PERM-P No. 949) was made to three Sakaguchi flasks containing 100 m1. of medium described in Example 1.

After 2 days cultivation, 10 mg. of 5-phenyl-7-trifluoromethyl-1,3-dihydro-2H-1,4-benzodiazepine-2-one dissolved in ethanol was added to each flask. Following 30 hours incubation the heavy growth which had developed was harvested by filtration, washed by 0.05 M phosphate buffer, pH 7.0, and suspended into 50 ml. of the same buffer but containing 0.1% of glucose.

Two ml. of sonicated suspension containing 100 mg. of 5 phenyl-7-trifluoromethyl-1,3-dihydro-2H-1,4-benzodiazepine-Z-one, which had been made from the substrate suspension in the phosphate buffer containing 0.1% glu cose by ultrasonicator, was added to the above mycerial suspension. The mixture was incubated at 28 C. on a Monod Shaker with addition of trace amounts of toluene.

After 20 hours incubation the reaction mixture was extracted by chloroform. The crude product was purified as in Example 2, by which yielded 3-hydroxy-5-phenyl-7- trifluoromethyl 1,3 dihydro-ZH-1,4-benzodiazepine-2- one melting at 189-191 C.

, slant was transferred to a 500 ml. Sakaguchi flask containing 100 ml. of the following medium; glucose 1 g., polypeptone 0.2 g., meat extract 0.1 g., yeast extract 0.1 g., tap water 100 ml., pH 7.0. The flask was incubated on a reciprocal shaker at 30 C. After 24 hours incubation, 100 mg. of 5-phenyl-7-nitro-1,3-dihydro-2H-1,4- benzodiazepine-2-one in 2.0 ml. of ethanol was added to the flask. After further 6 days incubation culture fluids were extracted with chloroform and extracts were evap rated to dryness under reduced pressure. The crude crystals, 97 mg., were purified as in Example 2, which by recrystallization from ethanol yielded 3-hydroxy-5-phenyl- 7-nitro-1,3-dihydro-2H-1,4-benzodiazepine 2 one. The identity of this compound was established by IR spectroscopy.

1 EXAMPLE 10 Streptomyces flavus ATCC 3369 was grown on the same medium described as Example 9. After 24 hours cultivation a mycelial transfer (2%) was made to each of ten new Sakaguchi flasks containing 100 ml. of the same medium as above. After good growth 100 ml. of -(2'-fluorophenyl)-7-chloro-1,3-dihydro-2H-1,4 benzodiazepine-Z-one in 2.0 ml. ethanol was added to each flask. Following 6 days cultivation culture fluids were extracted with chloroform and 755 mg. of crude crystals were obtained by the same procedure described in Example 1. The crude crystalswere purified by the similar procedure described in Example 2 by which yielded 3-hydroxy-5- (2'-fluorophenyl)-7-chloro-1,3-dihydro 2H 1,4-benzodiazepine-Z-one melting at 193-194 C. The identity of this compound was established by 'IR spectroscopy.

EXAMPLE 11 The fermentation was conducted with Streptomyces fradiae IFO 3360 as described in Example 1, except that the substrate 5-phenyl-7-methoxy-1,3-dihydro 2H 1,4- benzodiazepine-2-one was added.

The culture fluids were extracted by the method as in Example 1.

The crude products were purified as in Example 2, by which yielded 3-hydroxy-5-phenyl-7-methoxy 1,3 dihydro-2H-1,4-benzodiazepine-2-one.

The identity of this compound was established by IR spectroscopy.

1 2 EXAMPLE 12 The fermentation was conducted with Streptomyces griseus IFO 3102 as described in Example 1, except that the substrate 5-phenyl-7-methyl 1,3 dihydro 2H 1,4- benzodiazepine-Z-one was added.

The culture fluids were extracted by the method as in Example 1.

The crude crystals obtained were purified as in Example 2, by which yielded '3-hydroxy-5-phenyl-7-methyl-1,3- dihydro-2H-1,4-benzodiazepine-2-one.

The identity of this compound was established by IR spectroscopy.

EXAMPLE 13 Streptomyces griseochromogenes ATCC 14511 from an agar slant was transferred to a 500 ml. Sakaguchi flask containing m1. of the following medium glucose 1 g., soy bean meal 1.5 g., NaCl 0.5 g., tap water 100 ml., pH 7.0.

The flask was incubated on a reciprocal shaker at 28 C. After 24 hours incubation, a mycelial transfer (2%) was made to each of ten new flasks containing 100 ml. of the same medium as described above.

After additional 24 hours of incubation, 100 mg. of 5-phenyl-7,9-dichloro-1,3-dihydro 2H 1,4-benzodiazepine-2-one in 2.0 ml. of ethanol was added to each flask. After further 7 days incubation culture fluids were extracted with chloroform and extracts were evaporated to dryness under reduced pressure.

The residue was purified as in Example 2, by which yielded 3-hydroxy-5-phenyl-7,9-dichloro 1,3 dihydro- 2H-1,4-benzodiazepine-2-one.

The identity of this compound was established by IR spectroscopy.

EXAMPLE l4 Streptomyces benzodiazepinicus 8-145 (PERM-P No. 949) was grown on the same medium described in Example 13. After 24 hours incubation a mycelial transfer (2%) was made to each of ten new Sakaguchi flasks con taining 100 ml. of the same medium as above. After good growth 100 mg. of a compound selected from five different benzodiazepine Compounds A to E represented by the general formula:

H l H but different in R and R as shown in Table IV in 2.0 ml. of ethanol was added to each flask.

' TABLE IV A B c D E R. 01 01 F F CH3 R. 01 N02 01 N02 or After further 7 days incubation, culture fluids were extracted as described in Example 1 and purified as in Example 2, by which yielded 3-hydroxy derivatives of the substrate Compounds A to B, respectively.

The identity of those compounds was established by IR spectroscopy.

EXAMPLE 15 Streptomyces fimicarius 8-12 (FERM-P No. 945) was grown on the same medium described as Example 13. After 24 hours incubation a mycelial transfer (2%) was made to each of ten new flasks containing 100 ml. of the same medium as above. After good growth 100 mg. of a compound selected from four difierent benzodiazepine wherein C i ompounds A to D represented [by the general formula R 18 selected from the group consisting of hydrogen,

3* c carbamoyl, c c alkylcarbamoyl, c o alkyl, -0

H 5 alkenyl, C -C alkynyl, and C -C alkyl, C -C alkenyl and C -C alkynyl substituted by one to three radicals t 7 H selected from the group consisting of halogen, C -C cycloalkyl, aryl, a heterocyclic ring, aralkyl, acyl, carbamoyl, C -C alkylcarbamoyl, amino, C -C alkylamino, di C -C alkylamino and XR wherein X is a member of the group consisting of oxygen, sulfur,

but difi t in R1, R2 and R8 as shown in Table v in 'sulfonyl and sulfinyl, and R is a member of the group 2.0 ml. of ethanol was added to each flask. consisting of hydrogen, C -C alkyl, C -C alkoxy,

TABLE v A B c 1) R1 CH:

CHCH: HOCHa-CHz- CHlO-CHI CH:SCH:

R: H F H H R; c1 01 c1 01 After further 7 days incubation, culture fluids were ex- C -C alkyl, halo C -C alkyl, C -C alkenyl, C -C tracted as described in Example 1 and purified as in Exalkynyl, aryl, acyl, aralkyl, a heterocyclic ring, carample 2, by which yielded the compounds represented by bamoyl and CFC4 alkylcarbamoyl, the genual formula: R is selected from the group consisting of hydrogen, haloo gen and C -C alkyl, N OH R is selected from the group consisting of hydrogen, halogen, C -C alkyl and C -C alkoxy, R R is selected from the group consisting of halogen, tri- N fiuoromethyl, C -C alkoxy, C -C alkyl and nitro,

R is selected from the group consisting of hydrogen, halogen, C -C alkyl and C -C alkoxy and n is 0 or 1 which comprises fermenting a compound of Formula =1 but different in R R and R, as shownin Table VI. in the presence of a strain of Streptomyces having a 3-hy- TABLE VI Substrate A B c D Bi H HOCHr-CHr- H CHzO-CHP H CHIS"'OHT H X. I R: H H F F H H R; Cl 01 c1 01 c1 c1 01 01 The identity of those compounds was established by IR droxylating enzyme system to obtain a compound of the spectroscopy and thin layer chromatography. formula What we claim is: o 1. A method of causing the microbial transformation of a benzodiazepine compound of the formula E R1 0 l g R CH1 (oh R4 RI c=N- (o).

wherein R R R R R and n have the same meanings as above, or incubating a compound of Formula I (I) with resting cells of a strain of Streptomyces having a 1 3-hydroxylating enzyme system previously grown in a culture medium to obtain a compound of Formula II.

2. A method according to claim 1, wherein in the compound of Formula II, R is selected from the group consisting of hydrogen, C -C alkyl and cyclo C -C alkyl, R is selected from the group consisting of hydrogen and halogen, R is hydrogen, R is selected from the group consisting of halogen, trifiuoromethyl, C -C alkoxy, C -C alkyl and nitro, R is hydrogen and n is 0.

3. A method according to claim 1, wherein in the compound of Formula I, R is hydrogen, lower alkyl or cycloalkyl, R is selected from the group consisting of halogen and hydrogen, R is hydrogen, R is selected from the group consisting of halogen and nitro, R is hydrogen and n is 0, and in the compound of Formula H R R R R R and n have the same meanings as above.

4. A method according to claim 1, wherein the compound of Formula I is 1-cyclopropy1methyl-5-phenyl-7- chloro-1,3-dihydro-2H-1,4-benzodiazepine-2-one and the compounds of Formula II obtained are l-cyclopropylmethyl-3-hydroxy-5-phenyl-7 chloro 1,3 -dihydro-2H-1, 4-benzodiazepine-2-one and 3-hydroxy-5-phenyl-7-chloro- 1,3-dihydro-2H-1,4-benzodiazepine-2-one.

5. A method according to claim 1, wherein the compound of Formula I is 5-(2-fluorophenyl)-7-chloro-1,3- dihydro-2H-1,4-benzodiazepiue-2-one and the compound of Formula 11 obtained is 3-hydroxy-(5-(2-fluorophenyl)-7-chloro-1,3-dihydro-2H-1,4-benzodiazepine-2-one.

6. A method according to claim 1, wherein the compound of Formula I is 1-methyl-5-phenyl-7-ch1oro-1,3- dihydro-ZH-l,4-benzodiazepine-2-one and the compounds of Formula II obtained are 1-methyl-3-hydroxy-5-phenyl 7-chloro-1,3 dihydro 2H-l,4benzodiazepine-2-one and 3-hydroxy-5-phenyl-7-chloro 1,3 dihydro-2H-1,4-benzodiazepine-Z-one.

7. A method according to claim 1, wherein the compound of Formula I is 1-methyl-5-phenyl-7-nitro-1,3-dihydro-2H-1,4-benzodiazepine-2-one and the compounds of Formula II obtained are 1-methyl-3-hydroxy-S-phenyl- 7-nitro-1,3-dihydro-2H 1,4-benzodiazepine-2-one and 3 hydroxy-5-phenyl-7-nitro-1,3 dihydro 2H-1,4-benzodiazepine-2-one.

8. A method according to claim 1, wherein the compound of Formula I is l-methyl-S-phenyl-7-trifluoromethyl-1,3-dihydro-2H-1,4-benzodiazepine-2-one and the compounds of Formula H obtained are 1-methyl-3-hydroxy-5-phenyl-7-trifluoromethyl-1,3-dihydro-2H-1,4-ben- 10. A method according to claim 1, wherein the compound of Formula I is l-methyl-S-(2'-chlorophenyl)-7 chloro-1,3-dihydr o-2H-1,4-benzodiaZepine-2-one and the compounds of Formula II obtained are 1-methyl-3-hydroxy-5-(2'-chlorophenyl) 7 chloro-1,3-dihydro-2H- 1,4-benzodiazepine-2-one and 3-hydroxy-5 (2'-ch1orophenyl) 7 chloro-1,3-di-hydro-2H-1,4-benzodiazepine-2- one.

11. A method according to claim 1, wherein the compound of Formula I is 5-phenyl-7-trifluoromethyl-1,3-dihydro-2H-1,4-benzodiazepine-2-one and the compound of Formula II obtained is 3-hydroxy-5-phenyl-7-trifiuoro methyl-1,3-dihydro-2H-1,4-benzodiazepine-2-one.

12. A method according to claim 1, wherein the Streptomyces is Streptomyces albidoflavus ATCC15844.

13. A method according to claim 1, wherein the Streptomyces is Streptomyces flavus ATCC 3369.

14. A method according to claim 1, wherein the Streptomyces is Streptomyces griseachramogenes ATCC 14511.

15. A method according to claim 1, wherein-the Streptomyces is Streptomyces roseochromogenes IFO 3363.

16. A method according 'to claim 1, wherein the Streptomyces is Streptomyces fradiae TFO 3360.

17. A method according to claim 1, wherein the Streptomyces is Streptomyces griseus IFO 3102.

18. A method according to claim 1, wherein the Strepmyces is Streptomyces takarazukaensis 8-3 FERM-P No. 943.

19. A method according to claim 1, wherein the'Streptomyces is Strepwmlyces calvus S-4 PERM-P No. 944.

20. A method according to claim 1, wherein the'Streptomyces is Streptomyces fimicarius S-12 PERM-P No. 945.

21. A method according to claim 1, wherein the Streptomyces is Streptomyces chartreusis S-19 FERM-P No. 946.

22. A method according to claim 1, wherein the Streptomyces is Streptomyces transformans 8-60 FERM-P N0. 947.

23. A method according to claim 1, wherein the Streptomyces is Streptomyces glObisporus S- FERM-P No. 948.

24. A method according to claim 1, wherein the Streptomyces is Streptomyces benzodiazepinicus S'145 FER'M- P No. 949.

References Cited UNITED STATES PATENTS 3,453,179 7/1969 Greenspan et a1. 511; ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 

